The major objective of this proposal will be to elucidate the role of the carbohydrate moiety of fibrinogen, prothrombin and factor X in several of their biological properties: synthesis, secretion, functional activity, and in vivo and in vitro catabolism. The influence of the oligosaccharide on the synthesis and secretion of these proteins will be examined by culturing rabbit hepatocytes in the presence or absence of tunicamycin, a potent inhibitor of glycosylation. Fibrinogen, prothrombin and factor X synthesis will be determined by quantitation of 35S-methionine incorporated into the newly synthesized proteins. Degree of glycosylation will be determined by quantitation of 3H-mannose incorporation into proteins as measured by gel filtration. The functional properties of each protein will be tested by specific biological methods, and compared to those of their aglyco-counterparts. Aglycoprothrombin and aglycofactor X activation will be examined by identification of their activation products in fluorograms of SDS-PAGE gels. The in vivo catabolism of the aglycoproteins will be studied by infusing 35S-methionine labeled aglycofibrinogen or aglycoprothrombin into rabbits and comparing their plasma half-lives with those of the intact glycoproteins. The influence of the carbohydrate on the in vitro catabolism of these proteins will be assessed by examining the specific binding, internalization and degradation of 125I-labeled asialofibrinogen and asialoprothrombin by normal rabbit hepatocytes and human hepatoma cells (HepG2). The possible relationship between catabolism and synthesis of each particular protein will also be studied. Specifically, it will be determined whether degradation of the asialoderivative induces increased synthesis of its normal sialylated form. The binding of normal fibrinogen and its plasmic degradation fragments to rabbit hepatocytes and to human hepatoma cells will also be investigated. We will continue our studies characterizing the abnormal fibrinogen of liver disease. Fractionation of the abnormal fibrinogen in CON-A and determination of the carbohydrate composition of the glycopeptides by NMR and mass spectroscopy will elucidate the structure of the abnormal oligosaccharide and may uncover abnormal sugar linkages.